Difference between revisions of "Team:Uppsala/Zea-Strain"

Latest revision as of 02:15, 2 November 2017

Zeaxanthin

ZEAXANTHIN STRAIN

In our project we chose to concentrate on the pathway that leads from farnesyl pyrophosphate (FPP) to crocin (figure 1). The whole pathway consists of eight genes that code for eight enzymes which might make integrating all of the genes into a plasmid and keeping the plasmid in the bacteria more difficult. Dividing the pathway and integrating the part that leads from FPP to zeaxanthin into the chromosome would both give us a stable zeaxanthin-producing E. coli strain and make performing the remaining steps easier.

Figure 1. The pathway from farnesyl pyrophospate to zeaxanthin.

Resulting Zeaxanthin Producing E. coli Strain

We created a zeaxanthin producing E. coli strain using lambda red recombineering, with the whole pathway from FPP to zeaxanthin integrated into the chromosome (figure 2), which identified by the yellow pigment. All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain. After creating the zeaxanthin strain, we combined it with the plasmid containing the extended crocin pathway which gave us an E. coli strain including the entire production pathway from FPP to crocin. This will hopefully give other iGEM teams more freedom to work with and build on carotenoid pathways and make zeaxanthin more affordable to use in experiments.

Figure 2. Top: Wild-type *E. coli*. Bottom: Zeaxanthin producing *E. coli* strain with 5 genes inserted into the chromosome.

Zeaxanthin has previously been expressed in E. coli by iGEM teams using a plasmid. We decided to integrate this pathway into the chromosome using the Lambda red recombineering method. This would give our project several advantages such as releasing all the plasmid origins and cassettes which would make the insertion of the crocin pathway genes or any other genes of a pathway that originates from zeaxanthin easier. It would make the strain more stable because no constant selective pressure is needed and makes it possible to introduce larger constructs and longer pathways. This also means that there is no need for the use of antibiotics which makes the purification process easier, especially if the product is later used for nutritional purposes. And of course since the first step of our zeaxanthin pathway – farnesyl pyrophosphate – is endogenous to E. coli we would be able to express the whole pathway from farnesyl pyrophosphate to crocin with no need for costly intermediates. You can read about the design and details of the zeaxanthin strain production here.

We got our zeaxanthin producing strain with the whole pathway from FPP to zeaxanthin integrated into the chromosome! All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain (figure 3).

Figure 3. Left: Large scale expression of zeaxanthin from the zeaxanthin producing *E. coli* strain. Right: Extracted and purified zeaxanthin.

Besides observing the extracted zeaxanthin by eye we performed an absorbance measurement in the UV-Vis spectra (figure 4). Here we compared the absorbance spectra after two-phase extraction from the zeaxanthin strain, from wildtype E. coli and a zeaxanthin standard. The zeaxanthin strain had two peaks at 460 and 482 nm which were not present in wildtype E. coli. These peaks were also present in the standard, therefore we can conclude that our produced strain produces zeaxanthin. For the measurements the extracted compounds were dissolved in toluene.

Figure 4. Absorbance spectra for the extraction of zeaxanthin. MG1665 constitutes the negative control (the same extraction protocol on wildtype *E. coli*).

Figure 7. Thin layer chromatography for zeaxanthin producing strains with and without BioBricks in the crocin pathway, standards for crocetin, crocetin dialdehyde and negative control strain (MG1665). Samples from left to right: zeaxanthin producing strain, zeaxanthin producing strain with CaCCD2 (BBa_K2423005) plasmid, zeaxanthin producing strain with a combined CaCCD2 and CsADH2946 plasmid, crocetin standard, crocetin dialdehyde standard and MG1665 (wild type E. coli).

@@ Line 20: / Line 20: @@
        font-family: "Roboto Slab";
        font-size: 2.7vh;
+      line-height: 150%;
        color:#4D4D4D;
        text-align: justify;
+    }
+    .figtext {
+      font-family: "Roboto Slab";
+      font-size: 2vh;
+      font-weight: bold;
+      color:#4D4D4D;
+      text-align: justify;
+      padding-right: 20%;
+      padding-left: 20%;
+    }
+    .miniheader {
+      font-family: "Roboto Slab";
+      font-size: 2.7vh;
+      font-weight: bold;
+      color:#4D4D4D;
+    }
+    .picturerow {
+      display: inline-block;
+      height: 70vh;
+      display: auto;
      }
@@ Line 34: / Line 58: @@
    </div>
    <div class="row">
-     <div class="mainheader"> PROJECT DESCRIPTION </div>
+     <div class="mainheader"> ZEAXANTHIN STRAIN </div>
    </div>
    <div class="row">
      <div class="col-xs-4"></div>
      <div class="col-xs-4 textbox">
-       <img src="https://static.igem.org/mediawiki/2017/a/a3/Description_Yellow_Flask.png" style="margin: auto; display: block; width: 65%; height: auto; padding-top: 20%; padding-bottom: 20%;">
+       <img src="https://static.igem.org/mediawiki/2017/a/a3/Description_Yellow_Flask.png" style="margin: auto; display: block; width: 65%; height: auto; padding-top: 20%; padding-bottom: 10%;">
      </div>
      <div class="col-xs-4"></div>
    </div>
-  <div class="row">
-    <div class="mainheader"> ZEA-STRAIN </div>
-  </div>
-  <div class="row">
-    <div class="col-xs-12" style="height:60px;"></div>
-  </div>
    <div class="row text">
      <div class= "col-xs-1"></div>
      <div class= "col-xs-10">
-       <div style="padding-bottom:3%"> Zeaxanthin is a powerful antioxidant and one of the most common carotenoids found in nature [1]. A high concentration of zeaxanthin can be found in macula lutea which gives it a characteristic yellow color. There are multiple studies that suggest its important role in eye health [2]. Due to its commercial value there were many successful attempts to produce synthetic zeaxanthin. The Edinburgh iGem team from 2007 constructed a biobrick containing an operon
+       <div style="padding-bottom:3%;"> In our project we chose to concentrate on the pathway that leads from farnesyl pyrophosphate (FPP) to crocin (figure 1). The whole pathway consists of eight genes that code for eight enzymes which might make integrating all of the genes into a plasmid and keeping the plasmid in the bacteria more difficult. Dividing the pathway and integrating the part that leads from FPP to zeaxanthin into the chromosome would both give us a stable zeaxanthin-producing <i>E. coli</i> strain and make performing the remaining steps easier.
-with the genes necessary for zeaxanthin production (Fig.1) [3].</div>
+</div>
+<figure class="figure">
+      <img src="https://static.igem.org/mediawiki/2017/a/ab/Uppsala-ZeaPathway.png" style="display: block; margin: auto; width:60%; height: auto; padding-bottom:3%">
+        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 1. The pathway from farnesyl pyrophospate to zeaxanthin.
+</figcaption>
+      </figure>
+      <div class="miniheader"> Resulting Zeaxanthin Producing <i>E. coli</i> Strain </div>
+      <div style="padding-bottom:3%">We created a zeaxanthin producing <i>E. coli</i> strain using lambda red recombineering, with the whole pathway from FPP to zeaxanthin integrated into the chromosome (figure 2), which identified by the yellow pigment. All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain. After creating the zeaxanthin strain, we combined it with the plasmid containing the extended crocin pathway which gave us an <i>E. coli</i> strain including the entire production pathway from FPP to crocin. This will hopefully give other iGEM teams more freedom to work with and build on carotenoid pathways and make zeaxanthin more affordable to use in experiments.</div>
+      <figure class="figure">
+      <img src="https://static.igem.org/mediawiki/2017/1/18/Uppsala-ZeaPlate.png" style="display: block; margin: auto; width:40%; height: auto;"><br>
+        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 2. Top: Wild-type <i>E. coli</i>. Bottom: Zeaxanthin producing <i>E. coli</i> strain with 5 genes inserted into the chromosome.</figcaption>
+      </figure>
+      <div class="miniheader"></div>
+      <div style="padding-bottom:3%"> Zeaxanthin has previously been expressed in <i>E. coli</i> by iGEM teams using a plasmid. We decided to integrate this pathway into the chromosome using the <a href="https://2017.igem.org/Team:Uppsala/Experiments">Lambda red recombineering method</a>. This would give our project several advantages such as releasing all the plasmid origins and cassettes which would make the insertion of the crocin pathway genes or any other genes of a pathway that originates from zeaxanthin easier. It would make the strain more stable because no constant selective pressure is needed and makes it possible to introduce larger constructs and longer pathways. This also means that there is no need for the use of antibiotics which makes the purification process easier, especially if the product is later used for nutritional purposes. And of course since the first step of our zeaxanthin pathway – farnesyl pyrophosphate – is endogenous to <i>E. coli</i> we would be able to express the whole pathway from farnesyl pyrophosphate to crocin with no need for costly intermediates. You can read about the design and details of the zeaxanthin strain production <a href="https://2017.igem.org/Team:Uppsala/Design">here</a>.</div>
+      <div style="padding-bottom:3%"> <b>We got our zeaxanthin producing strain with the whole pathway from FPP to zeaxanthin integrated into the chromosome!</b> All of the steps were confirmed with PCR, gel electrophoresis and sequencing. We were able to extract and purify the expensive yellow zeaxanthin compound from our strain (figure 3).</div>
+      <figure class="figure" style="padding-left:20%">
+        <img src="https://static.igem.org/mediawiki/2017/0/0d/Uppsala-ZeaBottle.png" class="figure-img img-fluid picturerow">
+        <img src="https://static.igem.org/mediawiki/2017/b/bf/Uppsala-ZeaTube.png" class="figure-img img-fluid picturerow">
+        <figcaption class="figure-caption figtext" style="padding-bottom: 3%; padding-left:0%"> Figure 3. Left: Large scale expression of zeaxanthin from the zeaxanthin producing <i>E. coli</i> strain. Right: Extracted and purified zeaxanthin.</figcaption>
+      </figure>
+      <div style="padding-bottom:3%"> Besides observing the extracted zeaxanthin by eye we performed an absorbance measurement in the UV-Vis spectra (figure 4). Here we compared the absorbance spectra after <a href="https://2017.igem.org/Team:Uppsala/Experiments">two-phase extraction</a> from the zeaxanthin strain, from wildtype <i>E. coli</i> and a zeaxanthin standard. The zeaxanthin strain had two peaks at 460 and 482 nm which were not present in wildtype <i>E. coli</i>. These peaks were also present in the standard, therefore we can conclude that our produced strain produces zeaxanthin. For the measurements the extracted compounds were dissolved in toluene.</div>
        <figure class="figure">
-       <img src="https://static.igem.org/mediawiki/2017/d/d6/Uppsala-Zeaxanthin-Pathway.png" style="width:60%; height: auto; padding-bottom:3%">
+       <img src="https://static.igem.org/mediawiki/2017/c/c2/Extracted_Zeaxanthin.png" style="display: block; margin: auto; width:60%; height: auto;">
-         <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 1. Operon that was used by the Edinburgh iGEM team of 2007 with its respective gene products that are needed for the production for Zeaxanthin.</figcaption>
+        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 4. Absorbance spectra for the extraction of zeaxanthin. MG1665 constitutes the negative control (the same extraction protocol on wildtype <i>E. coli</i>).</figcaption>
+      <div class="miniheader">Combining the Zeaxanthin Producing Strain and the Crocin Pathway Enzymes</div>
+ <div style="padding-bottom:3%">
+After creating the zeaxanthin producing strain, we combined it with the plasmid containing the extended crocin pathway which gave us an <i>E. coli</i> strain including the entire production pathway from FPP to crocin. The <a href="https://2017.igem.org/Team:Uppsala/CrocinPathway">three enzyme BioBricks</a> BBa_K2423005, BBa_K2423007 and BBa_K2423008 in the zeaxanthin-crocin pathway were assembled to one plasmid (pSB1A3) using <a href="https://2017.igem.org/Team:Uppsala/Experiments">3A assembly</a> and was inserted into the zeaxanthin producing <i>E.coli</i> strain using <a href="https://2017.igem.org/Team:Uppsala/Experiments">electroporation</a>. The resulting plate can be seen in figure 5. </div>
+<figure class="figure">
+      <img src="https://static.igem.org/mediawiki/2017/3/3f/T--Uppsala--demonstrate_zeastrain.png" style="display: block; margin: auto; width:60%; height: auto;">
+         <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 5. Ampicilin plate with Zeaxanthin expressing <i>E. coli</i> strain transformed with pSB1A3 plasmid containing all three crocin pathway enzymes CaCCD2, CsADH2946 and UGTCs2.</figcaption>
        </figure>
+<div style="padding-bottom:3%">
+The color of the colonies changes slightly at each addition of another enzyme construct (another step in the crocin pathway). This is an indication that something is indeed happening with the bacterial pigment production when we introduce our pathway steps (figure 6) into the zeaxanthin strain. In the future it would be good to integrate the whole pathway from farnesyl pyrophosphate (FPP) to crocin into the chromosome for a stable crocin producing strain that does not require antibiotic selection which would make it easier to use as for example food coloring. </div>
-       <div style="padding-bottom:3%"> Later in 2013 this operon was used by the iGem team in Uppsala for their project [4]. Zeaxanthin is also a precursor to crocin - the substance our team was working on. Using this operon could potentially result in a crocin producing E.coli strain. Our problem was the size of this operon that if combined with additional genes of the crocin pathway could make this new strain unstable. That is why the Lambda red recombineering method was chosen for integration of the zeaxanthin pathway into E.coli chromosome instead of a plasmid. Our aim was to create a stable zeaxanthin producing strain with no need for selective pressure and then integrate a plasmid containing the crocin pathway.</div>
+<figure class="figure">
-       <div style="padding-bottom:3%"> Lambda red recombineering is based on homologous recombination which is mediated by bacteriophage lambda proteins [5]. In case of recombination with double-stranded DNA three different proteins: Exo, Gam and Beta derived from lambda red phage are required. Protein Exo degrades double-stranded linear DNA while Gam protects it from endogenous nucleases. Protein Beta is a key protein in the recombination process. It protects single-stranded DNA which was created by Exo and facilitates annealing to a complementary strand [6]. Lambda red recombination usually requires only about 35 base pairs of homology on both sides to work.[picture with lambda red enzyme functions?]*</div>
+       <img src="https://static.igem.org/mediawiki/2017/b/bc/Zea%2Bcaccd_and_zea%2Bcaccd%2Badh.png" style="display: block; margin: auto; width:60%; height: auto;"><br>
-      <div style="padding-bottom:3%"> Transduction is a well-established method which is used to insert DNA into bacterial strains. There are two types of outcomes following a transduction either the inserted DNA integrates into the chromosome of the host cell which is called the lysogenic life cycle or the bacteriophage mediate the lysis of the bacterial cell after a brief replication in an extrachromosomal form. For our experiment, Enterobacteria phage P1(P1) were used to perform transductions to assemble our genes. P1 is a temperate phage that replicates as an episomal replicon in the bacterial host until it lysis the cell. During the P1 replication bacterial genes can be inserted into the empty phage particles. For these Lambda Red mediated insertions, each bacterial strain had a Cat-sacB gene cassette in their chromosome which would be displaced by one of the previously mentioned genes. To get only one strain containing all the Crt genes two transductions had to be performed. One transduction to insert a new Cat-sacB gene cassette into an CrtI containing E. coli strain and a second transduction which would insert CrtZY together with a Trimethoprim resistance into a CrtEBI containing strain.[Flow chart?]*</div>
+        <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 6. Left: Chloramphenicol plate with Zeaxanthin expressing <i>E. coli</i> strain transformed with pSB1C3 plasmid containing CaCCD2. Right: Kanamycin plate with Zeaxanthin expressing <i>E. coli</i> strain transformed with pSB1K3 plasmid containing CaCCD2+CsADH2946 </figcaption>
+       </figure>
+<div style="padding-bottom:3%"> We wanted to analyze the compounds found after extraction of the zeaxanthin strains with and without plasmids from the crocin pathway. <a href="https://2017.igem.org/Team:Uppsala/Experiments">Thin layer chromatography (TLC)</a> was used to do this on a zeaxanthin strain, a zeaxanthin strain containing CaCCD2 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423005</a>), a zeaxanthin strain containing a plasmid with both CaCCD2 and CsADH2946, standards for crocetin and crocetin dialdehyde and a wild-type <i>E. coli</i> strain. As you can see from the resulting TLC plate in figure 7, many different pigments (represented by the bands) are present in the extract from the zeaxanthin strain. No pigment bands are present in the extract from the negative control strain (wild type <i>E. coli</i>), showing that the pigments in the zeaxanthin strain are precursors to zeaxanthin and zeaxanthin itself. This is consistent with the absorbance measurement where zeaxanthin was seen to be present in the extract (Figure 4.). Multiple pigments are also formed by the zeaxanthin + CaCCD2 and zeaxanthin + CaCCD2 + CsADH2946 strains (Figure 7.). Due to the mix of pathway pigments found in the samples, and limitations in resolution in the TLC, identification of specific pigment cannot be made from this data. In the future it would be very interesting to uniquelly identify the identity of the different pigments formed by our strains using a more informative method such as High-performance liquid chromatography-mass spectrometry (HPLC-MS).
+</div>
+      </figure>
+      </figure class="figure">
+      <img src="https://static.igem.org/mediawiki/2017/d/d0/TLC_Uppsala.png" style="display:block; margin: auto; width:30%; height: auto;">
+      <figcaption class="figure-caption figtext" style="padding-bottom: 2%; text-align:center;"> Figure 7. Thin layer chromatography for zeaxanthin producing strains with and without BioBricks in the crocin pathway, standards for crocetin, crocetin dialdehyde and negative control strain (MG1665). Samples from left to right: zeaxanthin producing strain, zeaxanthin producing strain with CaCCD2 (<a href="https://2017.igem.org/Team:Uppsala/Parts">BBa_K2423005</a>) plasmid, zeaxanthin producing strain with a combined CaCCD2 and CsADH2946 plasmid, crocetin standard, crocetin dialdehyde standard and MG1665 (wild type <i>E. coli</i>).</figcaption>
      </div>
      <div class= "col-xs-1"></div>
@@ Line 70: / Line 128: @@
      <div class="col-xs-12" style="height:40px;"></div>
    </div>
-  <div class="row text">
-    <div class= "col-xs-1"></div>
-    <div class= "col-xs-10">
-           <div style="padding-bottom: 1%;"> <b>References </b></div>
-           <div> (1) http://lpi.oregonstate.edu/ - micronutrient information center - dietary factors-carotenoids - 2017-09- 30 </div>
-           <div> (2) Eisenhauer, B.; Natoli, S.; Liew, G.; Flood, V.M. Lutein and zeaxanthin-food sources, bioavailability and dietary variety in age-related macular degeneration protection. Nutrients 2017, 9, 120. </div>
-           <div> (3) https://2007.igem.org/ - edinburgh -yoghurt- design - 2017-09- 29</div>
-           <div> (4) https://2013.igem.org/ - project -metabolic engineering - zeaxanthin -2017- 09-27</div>
-           <div> (5) Ellis, H. M., D. Yu, T. DiTizio &amp; D. L. Court, (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98: 6742-6746.</div>
-           <div style="padding-bottom: 2%;"> (6) Mosberg JA, Lajoie MJ, Church GM. (2010) Lambda red recombineering in Escherichia
-coli occurs through a fully single-stranded intermediate. Genetics 186: 791–799.</div>
-</div>
-    <div class= "col-xs-1"></div>
 </div>
@@ Line 101: / Line 141: @@
 </body>
 </html>
+{{Uppsala/Footer}}